转录因子ZNF582启动子高甲基化在鼻咽癌转移中的功能与机制研究

Distant metastasis is a major problem in the diagnosis and treatment of nasopharyngeal carcinoma (NPC). In recent years, it has been found that abnormal methylation of DNA plays an important role in tumorigenesis and development. Our previous studies showed that: a) The methylation level of ZNF582 gene in NPC tissues was significantly increased, while the mRNA expression level of ZNF582 was significantly decreased. Overexpression of ZNF582 significantly inhibited the migration and invasion of NPC cell lines. ChIP-seq combined with qPCR analysis showed that ZNF582 could promote the expression of Nectin-3 gene, and knockdown of Nectin-3 could reverse the inhibitory effect of ZNF582 on the migration and invasion of NPC cell lines. b) GEPIA database query found that ZNF582 was low expressed in various tumor tissues and predicted poor prognosis. Therefore, we hypothesized that ZNF582 may inhibit the metastasis of NPC by regulating the transcriptional expression of Nectin-3 gene and promoting cell movement. The purpose of this project is to: (1) to elucidate the role and molecular mechanism of ZNF582 in metastasis of NPC by in vivo and in vitro functional and biochemical experiments; (2) to illuminate the prognostic value of methylation of ZNF582 in metastasis of NPC by detecting in 300 cases of NPC tissues.

远处转移是目前鼻咽癌诊疗面临的主要难题，近年来发现DNA的异常甲基化在肿瘤发生发展中起着重要作用。我们的前期研究显示：①鼻咽癌组织中ZNF582基因的甲基化水平显著增高，而mRNA表达水平却显著降低；过表达ZNF582可显著抑制鼻咽癌细胞的迁移和侵袭；ChIP-seq结合qPCR分析发现ZNF582促进粘附分子Nectin-3基因表达，敲低Nectin-3可逆转ZNF582对鼻咽癌细胞迁移和侵袭的抑制作用。②GEPIA数据库查询发现ZNF582在多种肿瘤组织中低表达且预示不良预后。因此我们提出假说：ZNF582可能通过调控Nectin-3基因转录表达，从而影响细胞运动来抑制鼻咽癌转移。本项目拟在此基础上：①通过体内体外功能和生化实验阐明ZNF582在鼻咽癌转移中的作用和具体分子机制；②检测300例鼻咽癌组织中ZNF582启动子甲基化水平，明确其在鼻咽癌远处转移中的预后预测价值。

鼻咽癌在头颈部肿瘤中的转移率最高，高发于华南地区。放射治疗是目前鼻咽癌治疗的主要手段，而远处转移是鼻咽癌治疗失败和死亡的主要原因。因此，需要新的生物标志物来设计新的治疗策略以改善预后。ZNF582在多种肿瘤中异常表达。然而，他们在鼻咽癌中的表达水平和潜在功能及分子机制尚不清楚。.通过构建过表达和敲低表达模型，采用Transwell法检测ZNF582对鼻咽癌细胞的体外迁移侵袭能力的影响。采用裸鼠尾静脉-肺转移模型、足底腘-淋巴结转移模型移植瘤模型，评价ZNF582在体内对鼻咽癌细胞转移的影响。并利用高通量ChIP-seq测序结合体外生化实验探究ZNF582发挥功能的具体分子机制。.研究结果显示ZNF582在鼻咽癌细胞和组织中存在DNA高度甲基化，并导致其表达水平显著降低；功能研究发现ZNF582在体内外显著抑制鼻咽癌细胞的迁移侵袭，显著抑制细胞的肺转移和淋巴结转移。机制探究发现ZNF582可转录抑制和转录激活黏附分子Nectin-3和NRXN3的表达，而挽救实验表明回复Nectin-3或者抑制NRXN3的表达可逆转ZNF582对鼻咽癌细胞的侵袭转移的抑制作用，提示这两个下游基因是ZNF582的功能靶点。.我们的研究揭示ZNF582在鼻咽癌中扮演抑癌基因的角色，通过调控黏附分子的表达抑制鼻咽癌进展；低表达ZNF582的病人预后较差，提示靶向ZNF582可能是治疗鼻咽癌的新的策略，为鼻咽癌的个体化治疗提供了新的分子靶点。